Differential allele-specific expression (DASE) has been well described with the epigenetic phenomena of genomic imprinting and random monoallelic expression (RME). Recent studies from us and other groups have demonstrated that DASE is also relatively common among non-imprinted autosomal genes. Results from our previous studies have shown that DASE in BRCA1 expression was associated with an increased risk of developing breast cancer. As multiple genetic and epigenetic factors can contribute to DASE, DASE is a functional index for cis-acting regulatory variants and pathogenic mutations. These findings clearly support that DASE in cancer-associated genes could serve as a novel class of clinical biomarkers for cancer risk assessment, early detection, and treatment. The current gold standard platform for DASE measurement, allele-specific quantitative PCR (AS-qPCR), has several major limitations to making it impossible to map the observed DASE signals to individual cells and to apply for routine clinical practice. Although in situ hybridizaton (ISH) is routinely used in laboratory research and clinical settings, AS-RNA-ISH technology is considerably underdeveloped. RNAscope(R) is a novel RNA-ISH technology, which was recently developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. On the other hand, the recent development of toehold probe strategy helps to circumvent the pitfall of nonspecific hybridization created by traditional single allele-specific probe design. By seamlessly combining these two emerging technologies, we propose to develop a novel non-radioisotopic AS-RNA-ISH assay with high sensitivity and specificity, which is capable of evaluating both extreme and moderate DASE at individual cell levels using archived Formalin- Fixed Paraffin-Embedded (FFPE) tissues. To achieve this goal, we will first establish a new AS-RNA-ISH assay to detect truncating mutation-associated DASE in BRCA1 or BRCA2. Next we will validate this novel AS-RNA-ISH method with AS-qPCR using FFPE specimens from breast cancer patients. Results from our proposed study will help to establish a new AS-RNA-ISH method which will provide the capability of analyzing DASE in archived clinical specimens with high sensitivity and specificity, making this new approach a promising platform for translating DASE biomarkers into clinical use.